Introduction: Hydrogels are extensively used in 3D cell culture due to their biomimetic nature. We previously described a self-setting hydrogel based on silated hydroxypropylmethylcellulose (Si-HPMC) in which cross-linking of this polymer occurs by pH modification. This biomaterial is injectable, easy to handle and could provide an appropriate microenvironment for cell survival and differentiation.
Despite stem cell niche being characterized by a low oxygen content, anoxia at the core of cellularized scaffolds has long been associated with poor in vivo results of stem cell transplantation. Glucose and oxygen diffusion is therefore of outmost importance, since it influences cell metabolism and has a great impact on stem cell fate.
Hence, we aim to provide a detailed characterization of glucose and oxygen levels in Si-HPMC hydrogels. In this study, local oxygen and glucose concentrations were measured in cellularized constructs as a function of time and their impact on human adipose-derived stem cells (hASC) viability were evaluated.
Materials and Methods: Glucose measurements: Core glucose concentration was measured in situ with an implantable glucose sensor (WPI, USA). Glucose diffusion for different HPMC-Si hydrogel concentrations (from 0.2% to 4%) was measured on the core of ten millimeter-high acellular hydrogels following glucose concentration after addition of DMEM (5g/L glucose). Glucose consumption by hASC in HPMC-Si was also measured for different polymer concentrations and different cell densities.
Oxygen measurements: Core oxygen partial pressure was monitored in situ with a needle type oxygen microsensor (PreSens, Germany). For determination of oxygen content kinetics in acellular hydrogels, ten millimeter-high constructs were equilibrated at 5% oxygen and de-oxygenation profiles were recorded. In a separate experiment, hydrogels, seeded or not with (hASC) were incubated in normoxic conditions (20% O2) and core oxygen partial pressure was followed for different polymer concentrations and different cell densities.
Cell viability measurements: Staining of live hASCs inside hydrogel constructs was performed at 24, 48, 72 and 168 hours after cell seeding using a Calcein-AM Assay kit. Samples were collected from the center of constructs and imaged using confocal microscopy.
Results and Discussion: Although glucose diffusion was determined to be influenced by the polymer content, the highest polymer concentration (4%) seeded with 1 million cells/ml did not result in significant glucose depletion. On the other hand, increasing the cell density in these conditions provoked an acute glucose depletion as well as cell death.
Oxygen diffusion coefficient decreased from 1.2 mm2/h to 0.85 mm2/h as the polymer concentration increased from 1% to 4%, respectively. When cells were added to the constructs, cell viability also decreased in the same trend. In addition, an increase in cell density led to a significant decrease of the local oxygen concentration, with a complete depletion of oxygen observed with the highest cell density.
Conclusion: Although Si-HPMC hydrogels are mainly constituted of water that could favor solutes diffusion, we evidenced that high polymer concentrations impair oxygen diffusion while glucose concentration is mainly affected by cell density. In the next future, we will focus on adjusting these 2 parameters to design scaffolds with tunable diffusion properties. This could provide useful insights for the development of stem cell niche models.
Financial support : L. Figueiredo is a recipient of an Erasmus Mundus Doctorate fellowship (Nanofar)